Protein S is a key component for the regulation of hemostasis. Protein S serves as a cofactor for activated protein C, an enzyme responsible for the degradation of two protein elements of the blood coagulation pathway. Consequently, protein S through the action of protein C down-regulates the blood clotting process. Individuals deficient in functional protein S, either because of genetic or environmentally-acquired reasons, are at a higher risk of experiencing thrombotic disorders. Symptoms commonly associated with Protein S deficiency include thrombophlebitis, deep vein thrombosis, and pulmonary emboli. At the present time the genetic basis of protein S deficiency and its relationship to thrombosis are unknown. The long-term objective of the proposed research is to understand the genetic basis of protein S deficiency and thrombosis. This will be accomplished, in part, through the proposed studies, designed to provide a better understanding of the normal and abnormal protein S genes and the functional properties of the protein S products. Specific methods for achieving these goals include: a) cloning and characterization (by restriction endonuclease and DNA sequencing) of the normal human protein S gene and comparison to that from genetically protein S deficient individuals, b) chromosomal localization of the protein S gene by specific hybridization of protein S cDNA to mouse-human cell hybrids lacking different human chromosomes, c) quantitation of protein S messenger RNA levels in cells believed or suspected of participating in hemostasis and under the influence of hemostatic regulatory factors, and d) site-directed mutagenesis of protein S to address structure/function relationships, including the role of beta-hydroxyaspartic and gamma-carboxyglutamic acids, thrombin cleavage, and epidermal growth factor, propeptide and "Gla" domains in proteins S. The results may eventually lead to improved methods of diagnosis and therapy for patients having protein S deficiency and/or suffering from thrombotic disorders.